Monitoring of biotherapeutic purification by vibrational spectroscopy
File(s)
Author(s)
Beattie, James
Type
Thesis
Abstract
Monoclonal antibodies (mAbs) are used extensively as biotherapeutics (BTs) for a range of chronic and acute conditions. mAb treatments cost on average ∼$100 000 per year per patient, limiting their use. Protein A chromatography (PrAc) accounts for a substantial portion of downstream processing costs, primarily due to expensive resin and its degradation, resulting in reduced binding capacity. This research utilised vibrational spectroscopy to investigate these issues.
ATR-FTIR and Raman spectroscopy successfully assessed static binding capacity (SBC) of Protein A resin. Spatial location within the column affected resin performance, with the inlet showing greater binding capacity reduction compared to the outlet. Vibrational spectroscopic methods provided valuable chemical insights into protein conformation that traditional analysis methods lack.
In-situ ATR-FTIR spectroscopy revealed that changes in ligand density did not align with the reduction in binding capacity. Irreversibly bound contaminants were suggested as the primary cause of reduction in binding capacity. Confocal Raman spectroscopy demonstrated heterogeneous binding of BTs within resin beads, indicating reduced ligand accessibility in used resins. Spectral analysis of used resin samples without BTs loaded indicated the presence of Phenylalanine-rich co-eluting host cell protein species. This suggests irreversible binding of BTs and fouling mechanisms that reduce resin pore size.
Raman spectroscopy showed promise for monitoring dynamic binding capacity (DBC) of Protein A. High-throughput well plate Raman analysis proved as effective as industry-standard methods. By utilizing PLS analysis, Raman spectroscopy could quantify BTs not used in model training, reducing time and effort for each new BT. In-line Raman methodology successfully identified BTs in complex cell supernatant without separation or chemical labels, offering real-time monitoring potential.
in conclusion this thesis effectively used vibrational spectroscopy, specifically ATR-FTIR and Raman spectroscopy to characterise Protein A resin, elucidated binding capacity decay, and enabled DBC monitoring in BT downstream processing.
ATR-FTIR and Raman spectroscopy successfully assessed static binding capacity (SBC) of Protein A resin. Spatial location within the column affected resin performance, with the inlet showing greater binding capacity reduction compared to the outlet. Vibrational spectroscopic methods provided valuable chemical insights into protein conformation that traditional analysis methods lack.
In-situ ATR-FTIR spectroscopy revealed that changes in ligand density did not align with the reduction in binding capacity. Irreversibly bound contaminants were suggested as the primary cause of reduction in binding capacity. Confocal Raman spectroscopy demonstrated heterogeneous binding of BTs within resin beads, indicating reduced ligand accessibility in used resins. Spectral analysis of used resin samples without BTs loaded indicated the presence of Phenylalanine-rich co-eluting host cell protein species. This suggests irreversible binding of BTs and fouling mechanisms that reduce resin pore size.
Raman spectroscopy showed promise for monitoring dynamic binding capacity (DBC) of Protein A. High-throughput well plate Raman analysis proved as effective as industry-standard methods. By utilizing PLS analysis, Raman spectroscopy could quantify BTs not used in model training, reducing time and effort for each new BT. In-line Raman methodology successfully identified BTs in complex cell supernatant without separation or chemical labels, offering real-time monitoring potential.
in conclusion this thesis effectively used vibrational spectroscopy, specifically ATR-FTIR and Raman spectroscopy to characterise Protein A resin, elucidated binding capacity decay, and enabled DBC monitoring in BT downstream processing.
Version
Open Access
Date Issued
2023-04-05
Date Awarded
01/08/2023
License URL
Advisor
Byrne, Bernadette
Kazarian, Sergei G.
Rowland-jones, Ruth
Farys, Monika
Bettany, Hamish
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
GlaxoSmithKline
Grant Number
BB/S506965/1
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)